The p53 protein regulates cell proliferation by induction of growth arrest or apoptosis in response to DNA damage. A substantial proportion of cancer cells express wild-type p53, but it seems likely that in these cells p53 is inactivated. Among the known mechanisms by which cancer cells suppress functions of p53 is the regulation of p53 by the human double minute 2 oncoprotein (HDM2). The HDM2 protein binds to the trans-activation domain of p53 and inhibits its ability to activate transcription. Inhibition of p53 by HDM2 has been observed in tumors where gene amplification and other alterations can result in elevated HDM2 (one-third of soft tissue sarcomas). The disruption of the p53/HDM2 protein-protein interaction is therefore an attractive approach for cancer therapy because it provides the possibility to regulate the threshold of the p53 response with therapeutic agents. The crystal structure of the 109-residue amino-terminal domain of HDM2 (murine double minute 2 oncoprotein) bound to a 15-residue trans-activation domain peptide of p53 revealed that HDM2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic &#945;-helix. The interface relies on the steric complementarity between the HDM2 cleft and the hydrophobic face of the p53 &#945;-helix and, in particular, on a triad of p53 amino acids: Phe19, Trp23, and Leu26, which insert deep into the HDM2 cleft. Based on the 12-mer fragment of this p53 peptide (Gln16-Glu-Thr-Phe19-Ser-Asp-Leu-Trp-Lys-Leu-Leu-Pro27) and the 9-mer optimized peptide (Arg-Phe19-Met-Asp-Tyr-Trp-Glu-Gly-Leu26) we designed cyclic peptides, with side chains of residues 21(or 20) and 24 (or 25) linked together to form bridges (i, i+3 or i, i+4 type), to stabilize the &#945;-helical conformation. We used amide bond or thioether bond formation for peptide cyclization. By modification of amino acid residues and linker type we obtained several cyclic analogs. The conformational properties of these peptides were investigated using CD spectroscopy. Most of the cyclic peptides are more potent inhibitors of the p53/Hdm2 protein-protein interaction. During these studies we discovered the most potent peptide inhibitor containing only natural amino acids (IC50 392 nM). We also demonstrated that side-chain cyclization that stabilize helical conformation may be used to improve inhibitory potency of this peptide. We are going to improve the inhibitory potency of this peptide by optimizing the linker length and using unnatural amino acids. In collaboration with Prof. S. Wang from University of Michigan we also designed and synthesized fluorescent peptides with high content of unnatural amino acids (e.g. 5-Fam-&#946;Ala-&#946;Ala-Phe-Met-Aib-pTyr-(6-Cl-Trp)-Glu-Ac3c-Leu-Asn-Lys-NH2) with very high inhibitory potency against the p53/hdm2 protein-protein interaction (for the most potent peptide IC50 = 0.2 nM). These peptides were successfully used in development of the assay for testing the activity of small molecule inhibitors.